1. Field of the Invention
This invention pertains to the field of methods for concentrating target ligands and microorganisms from biological samples using binding moieties that are reversibly immobilized to magnetic particles. The methods provide highly sensitive means for concentrating and detecting target ligands and microorganisms that are present in a sample at very high dilution.
2. Background
The use of magnetic particles for concentrating target ligands, including microorganisms, from samples provides a convenient purification method that has several advantages over other methods such as gravitational or centrifugal separation. See, e.g., U.S. Pat. Nos. 4,628,037, 4,672,040, 4,695,392, and 4,695,393 for a description of magnetic particles and their use in separations. A target ligand that had been concentrated by use of magnetic beads would generally be detected while still associated with the beads. However, the presence of the magnetic beads associated with the concentrated target ligands can hinder detection of the target ligand. Nonspecific binding to the magnetic beads would limit the sensitivity of such methods. As a sufficient number of beads were added to a sample to bind all or most of the target ligand in the sample, the nonspecific binding to the beads increased, thus decreasing sensitivity. If fewer beads were used to decrease nonspecific binding, the sensitivity would also decrease because less than substantially all of the target ligand would be captured. Thus, the signal to noise ratio of these assays was relatively constant, placing a limit on sensitivity.
Thus, a need exists for a magnetic separation method for concentrating target ligands that is able to achieve a high degree of concentration of the target ligand. The present invention fulfills this and other needs.
The present invention provides methods, compositions, and kits for concentrating a target analyte in a test sample using magnetic beads. The method involves adding to the sample a target analyte binding moiety that specifically binds to the target analyte to form a target complex comprising the target analyte and target analyte binding moiety, and adding to the sample a magnetic bead to which is attached a capture moiety that specifically binds to the target analyte binding moiety to form a magnetic bead-bound target complex. A magnetic field is applied to the sample to collect the magnetic bead-bound target complex, after which the target complex is dissociated from the magnetic bead, thereby providing a concentrated target analyte. The target analyte binding moiety and the magnetic bead can be added to the sample simultaneously, or one can be added prior to the other.
In a preferred embodiment, the binding between the target analyte binding moiety and the target analyte is reversible under mild conditions, so that the target analyte binding moiety retains the ability to bind to the target analyte after dissociation. If the dissociation conditions also cause the target analyte binding moiety and the target analyte to dissociate, then following separation of the magnetic beads, the concentrated target analyte solution can be modified so that the target analyte binding moiety and the target analyte can immediately reassociate. The target analyte binding moiety and the target analyte typically remain in contact with each other during and after the dissociation step, and the target analyte can remain associated with the target analyte binding moiety throughout the dissociation step.
In another embodiment, the invention provides methods, compositions, and kits for detecting a target analyte in a sample. The methods involve the steps of a) adding to the sample a target analyte binding moiety that specifically binds to the target analyte, to form a target analyte/binding moiety complex; b) adding to the sample a magnetic bead to which is attached a capture moiety that specifically binds to the target analyte binding moiety, to form a complex comprising the target analyte binding moiety, the target analyte, and the magnetic bead; c) separating the complex from the sample by applying a magnetic force to the sample; d) dissociating the target analyte/binding moiety complex from the magnetic bead and removing the magnetic bead from the solution containing the binding peptide-target analyte complex; and e) detecting the presence of the target analyte/binding moiety complex.
In one embodiment, the detection step involves an immunoassay. The immunoassay can be performed by applying the solution containing the target analyte binding moiety-target analyte complex to a solid support upon which is immobilized an anchor moiety that specifically binds to an epitope of the target analyte; applying a detection moiety that specifically binds to a hapten present on the target analyte binding moiety; and detecting the label. The target analyte epitope to which the target analyte binding moiety binds can be the same as or different than the epitope to which the anchor moiety binds.
The invention also provides kits and devices for detecting a target analyte in a sample. The kits can include a target analyte binding moiety that is capable of specifically binding the target analyte and a plurality of magnetically responsive particles to which are attached a plurality of capture moieties that are capable of reversibly binding the target analyte binding moiety. The target analyte binding moiety can include a molecular tag to which the capture moiety binds. The kits can also include a detection moiety that is capable of binding to the molecular tag, control antigens, and the like.